Singh Deepak Kumar, IJSRR 2018, 7(3), 1990-2036 Review article Available online www.ijsrr.org ISSN: 2279–0543

International Journal of Scientific Research and Reviews

In Vitro Propagation of Orchids For Their Conservation: A Critical Review

Deepak Kumar Singh

Department of Botany, University of Delhi, Delhi 110007, E-mail: [email protected]

ABSTRACT Orchids are the most pampered, gorgeous and peculiar with implausible range of diversity. Their magnificent flower with stunning colour, glamorous shape and long lasting features make them commercially important.Inspite of having spectacular advancement in in-vitro micropropagation of orchids, problems frequently encountered have been exudation of large quantity of phenolics, choice of appropriate explants, shortage of efficient methods for seed germination, and seedling death during inoculation. All orchids have been listed in Appendix II of CITES and some even have been included in Appendix I. Present review makes an effort to bring together some recent studies on orchids via seeds, rhizomes, shoots tip, internodes, pseudobulbs, PLBs, leaves, roots, nodes as explants . These reported protocols, after initial testing their reliableability and efficiency can possibly be used for large scale mass multiplication along with ex vitro establishment of rare, threatened and endangered orchids to meet the horticultural, floricultural market demand. KEYWORDS: Protocorm like bodies, Orchids, Seed germination, Conservation, Mass propagation.

*Corresponding author Deepak Kumar Singh Department of Botany, University of Delhi, Delhi 110007, India Corresponding author E-mail: [email protected]

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Singh Deepak Kumar, IJSRR 2018, 7(3), 1990-2036 ORCHIDS: A BRIEF INTRODUCTION Orchid’s flowers are one of the most beautiful, peculiar and fascinating creations of God. The term ‘Orchid’ derives its origin from the Greek word ‘Orchis’; meaning testicles 1. Owing to the shape of their pseudobulbs resembling testicles, these were named as orchids. The term orchid was first used by the famous Greek philosopher, Theophrastus (372-286 B.C) in his book “de Historia Plantarum”. He also highlighted therapeutic significance of orchids in his book “Enquiry into Plants” 2 . The orchid family, , is one of the largest, most evolved and diverse families of flowering plants. It comprises 17000 to 35000 species belonging to 750 to 850 genera 3. About 1300 species are estimated to occur in India 3, 4. The latest estimate in terms of the numerical strength of the members of this family is expected to fall somewhere close to 20,000 species 5. A new estimate made for Appendix 2 of CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) is 3 about 19,500 species . Orchids are cosmopolitan in distribution, occurring almost in all habitats including glaciers and dry desert 6,7. Their extraordinary diversity predominates in tropical and subtropical zones 6,4 . About 73% of orchids are epiphytes and rest are lithophytes, semi-terrestrial and true terrestrial 3. They use other plants (trees) merely for support and space. Monopodial (single stem) and Sympodial (multiple stems) are the two major growth patterns found among orchids. Indeterminate growth of monopodial stem produces leaves but lacks rhizome 8. Orchids belonging to this category are Phalaenopsis, Vanda sp., Vanilla sp. and so on 9,10. The sympodial orchids possess storage organs known as pseudobulbs which act as reservoirs of food and water. This habit appears as successive growth, each originating from the base of preceding one, e.g. Paphiopedium sp., Oncidium sp., Dendrobium sp., Cattleya sp., sp., Arundina sp., Phaius sp. and Anoectochilussp.etc.8,10. Orchids reproduce by means of seeds with pods or capsules being the fruiting body, each of which contains millions of microscopic, which disperse like spores or dust particles, contain neither endosperm nor fully differentiated embryo11-14. Despite production of seeds in large numbers, the plants produced are limited because of the low survival rate of seeds and high rate of mortality of seedlings12-16. Cotyledon, radicle and plumule are almost absent except in few species, such as, Sorbralea macrantha and Bletilla hyacynthina, which have well differentiated embryos and rudimentary cotyledons 9.

MEDICINAL ORCHIDS A detailed literature survey has revealed that 209 species of orchids are used for the treatment of one or the other ailments afflicting human beings (Table 1). Tubers are the most commonly used organs for therapeutic purposes followed by whole plants, roots, pseudobulbs and rhizomes (Fig. 1).

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Figure 1: Parts of orchid plants used as herbals.

Table1 - List of orchids used for various medicinal purposes as per quoted literature. S. No. Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) 1. carinata Rasna (H), Small Root, leaf The root paste is used in 17 (Griff.) Panigrahi. Warty Acampe (E) scorpion and snake bite, rheumatism and uterine diseases. 2. Aerides crispum - Whole plant Cure ear pain and deafness 18 Lindl 3. Acampe papillosa Small Warty Acampe Root It is useful in poisonous 19 (Lindli.) Lindl. (E) infections, and fever 4. Acampe praemorsa Acampe Orchid (E) Root Root paste of Acampe 20 (Roxb.) Blatt. & McCann praemorsa and Asparagus recemosus are taken empty stomach to cure arthritis. 5. Acampe wightiana Root The plant is used to make 21 Lindl. - tonic and also useful in cold and cough. 6. Aerides multiflorum - Whole plant It showed antibacterial activity 22 Roxb. against Salmonella auereus and Klebsiella pneumonia. 7. Aerides odorata - Root, leaf Reduces joint pain and 17

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) Lour. swelling. The leaf juice is taken to cure tuberculosis. 8. Anoecetochilus formosanus Jewel Orchid (E) Tuber It is useful in diabetes, fever 23,24 Hayata. and liver spleen disorder. 9. Anoectochilus roxburghii Roxburgh’s Whole plant Treatment of fever, 25 (Wall.) Lindl. Anoectochilus (E) pleurodynia, snake bike, lung and liver disease, hypertension. 10. Arundina graminifolia Bamboo Orchid, Rhizome Root decoction is used as 26 (D.Don) Hochr. Tapah Weed, Kinta pain killer. Weed, Bird Orchid (H) 11. Bletilla formosana The Beautiful Bletilla Tuber Used for the treatment of 23 (Hayata) Schltr. (E) lung, liver and stomach disorder. 12. Bletilla striata Chinese ground Tuber Antibacterial and anti- 27,28 (Thunb.) Rchb. f. orchid, Hardy orchid inflammatory. 13. Bletia hyacinthina Hyacinth Orchid Tuber Beneficial in tuberculosis, 29 (Wild) R.Br. cracked skin, ulcers and breast cancer. 14. Brachycortis obcordata Heart-Shaped Root Use as tonic with milk, cure 30 (Lindl.) Summerh. Brachycorythis dysentery 15. careyanum Carey’s Leaves, Cause abortion, used in burns -do- (Hook.) Sprengel Bulbophyllum pseudobulb 16. Bulbophyllum cariniflorum Keeled Flower Root Induce abortion within 2-3 17 Rchb. F. Bulbophyllum (E) month of pregnancy 17. Bulbophyllum kwangtungense The Kwangtung Tuber Treat pulmonary 31 Schltr. Bulbophyllum (E) Tuberculosis, reduce fever and promote the production of body liquid 18. Bulbophyllum leopardinum The Leopard Spotted Whole plant Decoction used in burns 30 (Wall.) Lindl. Bulbophyllum 19. Bulbophyllum lilacinum Ridl. The Lilac Pseudobulbs Fluid of pseudobulb with 4 Bulbophyllum (E) water keep the body fresh and remove tiredness. 20. Bulbophyllum odoratissimum The Fragrant Whole plant Treat tuberculosis, chronic 32 (Sm.) Lindl. Bulbophyllum (E) inflammation and fracture. 21. Bulbophyllum neilgherrense The Nilgiri Mountain Pseudobulbs Pseudobulp juice restore 4

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) Wight Bulbophyllum (E) youthness and act as antiageing medicine. 22. Bulbophyllum umbellatum The Umbrella Whole plant Enhance congenity 30 Lindl. Bulbophyllum 23. Calanthe discolor Ground Orchid (E) Whole plant Hair restoring. 33 Lindl. 24. Calanthe liukiuensis - Whole plant Hair restoring. 33 Schltr. 25. Calanthe plantaginea The Plantago-Like Rhizome Used as tonic and 30 Lindl. Calanthe aphrodisiac 26. Calanthe puberula The Hairy Calanthe Rhizome Used with milk as tonic -do- Lindl. 27. Calanthe sylvatica The Forest-Dwelling Flower Cures nose bleeding -do- (Thou) Lindl. Calanthe 28. Calanthe tricarinata Monkey Orchid, Leaf, Leaves decoction applied on -do- Lindl Hardy Calanthe Pseudobulbs sores and eczema. Orchid Pseudobulbs having aphrodisiac properties 29. Catasetum barbatum The Bearded Whole plant Febrifuge, anti- 34 Lindl. Catasetum (E) inflammatory. 30. Cephalanceropsis gracillis - Whole plant Suppress cancer 31 (Lindl.) 31. Cephalanthera longifolia Narrow-leaved Rhizome Heals wounds. Used as 30 K. Fritsch Helleborine or appetizer and tonic. Sword-leaved Helleborine 32. Cleisostoma williamsonii Williamson’s Whole plant Plant’s paste is used as 19 (Rchb.f.) Garay. Cleisostom (E) astringent. 33. corymbosa Lindl. The Umbrella-Like Pseudobulb Pseudobulb juice used in 35 Coelogyne (E) healing wounds 34. Coelogyne cristata Crested Coelogyne Pseudobulb Heals wounds 17 Lindl. (E), Gondya (H) 35. Coelogyne flaccida The Loose Coelogyne Pseudobulb Useful in headache, fever 30 Lindl. and constipation 36. Coelogyne fuscescens Ocher Yellow Pseudobulb Treatment of stomachache. 36 Lindl. Coelogyne (E) 37. Coelogyne gardneriana Lindl. Gardner’s Neogyna Whole plant Antibacterial against 37 (E) klebsiella pneumoniae and

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) E.coli. 38. Coelogyne nitida The Shining Pseudobulb Paste is useful in fever, 30 (Wall. ex Lindl) D. Don. Coelogyne headache and burn. 39. Coelogyne ovalis The Oval Coelogyne Pseudobulb Aphrodisiac -do- Lindl. 40. Coelogyne prolifera Prolific Coelogyne, Pseudobulb Relieve fever, headache and -do- Lindl. Yellowish Coelogyne backache 41. Coelogyne stricta The Rigid Coelogyne Pseudobulbs Relieves fever and headache -do- (D. Don) Schltr 42. Coelogyne viride - Rhizome Memory deficits. 38 (L.) 43. Coelogyne punctulata Spotted Coelogyne Pseudobulb Pseudobulb powder is used -do- Lindl. (E) in burn injury and healing wounds. 44. Conchidium muscicola - Whole plant Useful in repiratory, cardiac 30 (Lindl.) Lindl. and nervous disorders 45. Corallorhiza maculate Spotted Coral Root Roots, stalks Dried stem is used in making -do- Raf. (E) tea and treats pneumonia patients 46. Coryborkis veratrifolia White Cinnamon Leaf Leaf juice is used to treat -do- (Reinw.) Blume Orchid (E) fever. 47. Cremastra appendiculata Bulbs It is associated with liver, 39 (D.Don) Makino spleen and stomach - meridians. Fight tumors and skin lesions. 48. Crepidium acuminatum Jivak (H) Rhizome, Treats weakness, fever, 30 (D. Don) Szlach root, tuberculosis. and bronchitis. psudobulb 49. Cymbidium devonianium - Whole plant Treats cough and cold -do- Lindl. ex Paxton 50. Cymbidium elegans The Elegant Leaves, Used for healing wounds -do- Lindl. Cymbidium Pseudobulbs, roots 51. Cymbidium iridioides The Iris-Like Leaves, Used as tonic and stop -do- D. Don Cymbidium Pseudobulbs, bleeding. roots 52. Cymbidium goeringii Hardy Cynbidium Whole plant Shows diuretic activities. 40 (Rchb.f.) Orchid (E)

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) 53. Cymbidium macrorhizon Large Root Rhizome Used as diaphoretic and 41 Lindl. Cymbidium (E) febrifuge. 54. Cymbidium aloifolium Leaf Root, leaf, Reduce paralysis. 17 (L) Sw. Cymbidium (E) whole plant 55. Cymbidium ensifolium Cymbidium With The Rhizome Decoction from rhizome cure 19 (L) Sw. Sword Shaped Leaves gonorrhoea and eye sores. (E) 56. Cymbidium giganteum wall ex The Iris-Like Leaf Leaf juice has blood clotting 42 Lindl. Cymbidium (E) properties 57. Cymbidium longifolium - Pseudobulb Used as demulcent. 35 D.Don 58. Cypripedium calceolus Yellow lady slipper Root, Useful in headaches, 43 L. (E) rhizome diabetes, dysentery, paralysis etc. 59. Cypripedium cordigerum Heart-shaped Slipper Roots Used as tonic 30 D. Don Orchid 60. Cypripedium elagans Rchb.f. Elegant Slipper Root Used in epilepsy, 19 Orchid (E) rheumatism, madness and hysteria. 61. Cypripedium himalaicum Himalayan Slipper Whole plant Cures heart,chest, stone and 30 (Rolfe) Kranzl. Orchid urinary disorders. 62. Cypripedium macranthos The Large Flowered Rhizome Used for skin diseases. 44 Cypripedium (E) 63. Cypripedium parviflorum Lesser Yellow Lady’s Rhizome Treats various disorders like 45 Salisbury Slipper, Smaller anxiety, fever, headache, Yellow Lady’s tension, insomnia, pain of Slipper (E) menstruation and child birth. 64. Cypripedium pubescens Wild. Greater yellow lady’s Root It is used in diarrhea, 6 slipper (E) dysentery, malnutrition, diabetes, impotence, headache. 65. Dactylorhiza hatagirea Hathajari, Hathpanja Root, Tubers used in tonic and 2 (D.Don) Soo. (H) rhizome aphrodisiac. 66. Dactylorhiza maculate The Spotted Tuber The plant has aphrodisiac 46 ( L.) Soo Dactylorhiza (E) properties. 67. Dactylorhiza purpurella Northern Marsh Tuber It has antiageing and -do- (Stephen. & Stephen.) Soo. Orchid (E) aphrodisiac properties. 68. Dendrobium alpestre The Mountain Living Pseudobulb Used to treat pimple and 41

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) Royle Dendrobium (E) other skin problem. 69. Dendrobium amoenum Wall. The Lovely Leaf Skin diseases 47 ex Lindl. Dendrobium (E) 70. Dendrobium aurantiacum The Yellow Flowered Leaf Diabetes 48 Rchb.f. Dendrobium (E) 71. Dendrobium candidum Wall. The White Leaf Diabetes 49 ex Lindl. Dendrobium (E) 72. Dendrobium crepidatum The Shoe-lipped Pseudobulb Paste applied in dislocation 30 Griff. Dendrobium and fracture of the bone. 73. Dendrobium chrysanthum Golden Flowered Leaf Antipyretic, eyes-benefiting, 50 Wall. Dendrobium (E) immunoregulatory purposes, skin diseases 74. Dendrobium crumenatum Sw. The Pigeon Orchid Leaf Beneficial in pimples 19 (E) 75. Dendrobium densifiorum The Densely Leaf Promotes the production of 51 Lindl. ex Wall. Flowered body fluid. Dendrobium (E) 76. Dendrobium densiflorum Pineapple Orchid Psudobulbs Remove pimples and cures 30 Lindl. other skin problem. 77. Dendrobium eriaeflorum The Eria-Like Psudobulbs Used as tonic and even -do- Griff. Flowered applied in bone fracture. Dendrobium 78. Dendrobium farmeri Farmer’s Dendrobium Whole plant Antibacterial activity against 37 Paxton (E) Klebsiella pneumoniae,E.coli and Salmonella typhi. 79. Dendrobium fimbriatum The Fringe-Lipped Leaf Leaf paste applied on 52 Hook. Dendrobium (E) fracturated area to set bone. 80. Dendrobium heterocarpum Noble Dendrobium Psudobulbs Paste applied in dislocation 30 Wall.ex Lindl. and fracture of the bone.

81. Dendrobium herbaceum Grassy Dendrobium Leaf Paste of fresh leaves applied 17 Lindl. (E) on wound and treats syphills. 82. Dendrobium loddigesii Rdfe. Loddiges’ Leaf Used as tonic to nourish the 53 Dendrobium (E) stomach, replenish body fluid, and reduce fever along wtih anticancer properties. 83. Dendrobium longicornu - Whole plant Relieve cough and fever 30 Lindl.

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) 84. Dendrobium macraei Tubers Used in tonic preparation. 54 Auct. Jivanti(H) 85. Dendrobium macrostachyum Leafless Dendrobium Tender shoot Tender tip juice is used as an 55 Lind. (E) tip ear drops for earache. 86. Dendrobium monoiliforme The Necklace-Shaped Stems Tonic, antipyretic, 56 Dendrobium (E) aphrodisiac, analgesic. 87. Dendrobium monticola The Mountain Living Pseudobulb Useful in pimples and skin 19 Hunt & Stummerh. Dendrobium (E) eruption. 88. Dendrobium moschtum Lindl. The Musky-smelling Pseudobulb Treat dislocated and 30 Dendrobium fractured bones 89. Dendrobium nobile Noble Dendrobium( Pseudobulb Plant is used in the treatment 57,58 Lindl. E) seed of pulmonary tuberculosis, dyspepsia,fever and anorexia. 90. Dendrobium normale - Whole plant Entire plant have aphrodisiac 4 Falc. and tonic properties. 91. Dendrobium ovatum Green Lipped Whole plant Useful in stomachache and 19 (Wild.) Kranzl. Dendrobium (E) constipation. 92. Dendrobium pumiluim Roxb. The Broad-Stemmed Whole plant The Malays use it as a drug 59 Dendrobium (E) in dropsy. 93. Dendrobium tosaense Makino - Leaf Treatment of anxiety and 60 panic. 94. Dendrobium transparens The Translucent Pseudobulb Treat dislocated and 30 Wall. ex Lindl. Dendrobiu fractured bones 95. Desmotrichum fimbriatum - Whole plant Cure disorder of bile, blood 59 Blume and phlegm. Helpful in treatment of snake bite, scorpion sting and even used as tonic in debility due to seminal losses. 96. Dienia cylindrostycha Pseudobulb Used as tonic 30 Lindl. 97. Ephemerantha lonchophylla - Stems It is used as tonic to nourish 61 (Hook . F.) Hunt & Summerch the stomach, promote the production of body fluid, and reduce fever. 98. Epipactis helleborine Bastard Hellebore, Root, Roots of this plant are 2 (L.) Crantz. Broadleaf Helleborine, rhizome medicinal which cure Common Helleborine insanity.

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) (E) 99. Epipactis latifolia Broad-helleborine, Rhizome Rhizome is regarded as 4 Wall. Helleborine orchid aphrodisiac and is used to treat (E) nervous disorder. Infusion of leaves is useful in case of intermittent fever. 100. Epidendrum mosenii - Stem Analgesic activity 52 Barb. Rodr. 101. Epidendrum rigidum Rigid star orchid (E) Stem Replenish body fluid 63 Jacq. 102. Eria bambusifolia The Bamboo-Like Whole plant The plant is used to cure 64 Lindl. Leafed Eria (E) hyper acidity and other stomach disorders 103. Eria muscicola The Moss-Growing Pseudobulb It is used to treat chest, heart, 65 (Lindl.) Lindl. Eria (E) lung, eye, ear and mental problems 104. Eria pannea The Flag Eria (E) Root, leaf Decoction of leaves and roots 19 Lindl. are used in boneache. 105. Eria spicata The Spicate Eria (E) Stem Stem paste is used to cure 35 (D.Don.) Hand Mazz headache and stomach disorder. 106. Eria pubescens - Fluids extracted from 4 Lindl. pseudobulb mixed with water and taken to increase the sexual strength. 107. campestris Salep (E), Salam (H) Tuber Blood purifier, demulcent 59 Wall. anthemnitic 108. Eulophia dabia Salep misri (H) Salam Tuber Tubers used in stomach 2 (D.Don) Hochr. misiri (H) tonic, aphrodisiac and blood purifier during heart problem. 109. Eulophia epidendraea The Epidendrum-Like Tuber The tubers are used as 19 (J.König ex Retz.) Eulophia (E) vermifuge and to treat C.E.C.Fisch. anorexia and anthrax. 110. Eulophia graminea Grass Eulophia (E) Tuber Tuber’s decoction are used as 66 Lindl. vermifuge. 111. Eulophia herbacea - Tuber It is supposed to be a kind of 19 Lindl. salep and tonic.

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) 112. Eulophia nuda Whitton root, Salep Tuber Demulcent, anthemnintic and 59 L. (E) helpful in cardiovascular diseases. 113. Eulophia ochreata Golden-Yellow Tuber Useful in male sterility, 67 Lindl. Eulophia (E) sexual impotency, vigour and to some extent show aphrodisiac properties. 114. Eulophia pratensis The Spectacular Tuber Tubers are used externally as 59 Lindl. Eulophia(E) well as internally to remove scrofulous gland in the neck. 115. Eulophia spectabilis The Spectacular Leaf Aphrodisiac and leaf 17 (Dnnst.) Suesh Eulophia (E) decoction is also used against vermifuge. 116. Flickingeria fugax Whole plant Used as tonic 30 (Rchb. f.) Seidenf. 117. Flichingeria macraei (Lindl.) Macrae’s Flickingeria Pseudobulb Extracted juice of pseudobulb 17 Sidenf. (E) should be taken twice a day for 21 days to cure skin allergy and even applied on an affected part to cure eczema. 118. Galeola foliate Great Climbing Stems Treatment of some infections 68 (F.Muell.) Orchid (E) 119. Galeris strachaeyi - Tuber Cure headache and even used 30 (Hook. f.) P. F. Hunt as tonic

120. Gastrodia elata Gastrodia (E) Whole plant Treatment of convulsive 29 Blume. diseases such as epilepsy 121. Gastrodia orobanchoides Tuber Tubers are edible and sweet 4 (Flac.) Benth. - in taste . 122. Geodorum densiflorum (Lam.) Nodding Swamp Root, tuber Fresh root paste with honey 17 Schltr. Orchid, Shepherds regulate menstrual cycle, Crook Orchid (E) snake bite, cuts and wounds. 123. Geodorum recurvum (Roxb.) The Bent Geodorum Tuber Decoction of dried tuber are -do- Alston (E) used to cure malaria and suppress tumors. 124. Goodyera repen Creeping Rattlesnake Cures appetite, stomachache, 4 (L.) R.Br. Plantain, Lesser cold, kidney, stomach Rattlesnake Plantain disorder.

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) (E) 125. Goodyera schlechtendaliana - Whole plant Tonic for internal injuries 69 Rchb. f. and to improve circulation. 126. Gymnadenia conopsea Fragrant Orchid (E) Tuber It is used as aphrodisiac 70 (L.) R.Br. 127. Gymnadenia orchidis Himalayan Fragrant Roots, Heals wound and even used 30 Lindl. Orchid Pseudobulbs in urinary and liver disorder

128. Habenaria acuminata The Acuminate Tuber The tubers are used as tonic. 2 Lindl. Habenaria(E) 129. Habenaria commelinifolia Commelina-Leaf Root 6-8 drops of this roots 17 (Roxb.) Wall.ex Lindl. Habenaria( E) decoction administered orally on an empty stomach for 10 days to cure spermatorrhea and urinary trouble. 130. Habenaria crinifera The Hair Carrying Tuber Cure headache 71 Lindl. Habenaria (E) 131. Habenaria edgeworthii Hook. Vridhi (H) Tuber Rejuvenator, spermopiotic 2,72 F.ex Collett. Edgeworth’s and even regarded as tonic Habenaria (E) and blood purifier. 132. Habenaria foliosa The Leafy Habenaria Tuber Plant is used medicinally by 21 (Sw.) Rchb. f (E) the Zulus (). 133. Habenaria furcifera The Fork-Carrying Tuber Ointment for cuts, wounds 55 Lindl. Habenaria (E) and poisonous bites. 134. Habenaria goodyeroides The Goodyera-Like Tuber The tuber is used as tonic. 2 D.Don Peristylus (E) 135. Habenaria griffithii Griffith’s Diphyllax Tuber The tuber is used as tonic. -do- Hook.f.(D.Don) (E) 136. Habenaria hollandiana Sant. - Tuber Fresh plant paste is aaplied 55 externally for scorpion sting and for infested sores. 137. Habenaria intermedia D.Don Ridhi (H) Tuber Cooling, spermopiotic, 2,72 Intermediate rejuvenator and even used as Habenaia (E) tonic, cure various blood diseases. 138. Habenaria longicorniculata The Small Horned Tuber Tubers decocotion with 17 Graham Habenaria (E) turmeric powder applied externally on affected part to

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) cure leucoderma. 139. Habenaria marginata Coleb. Golden Yellow Tuber Treatment of malignant ulcer -do- Habenaria (E) . 140. Habenaria pectinata Comb Habenaria (E) Leaf, tuber The leaves are crushed and 72 D.Don applied in case of snake bites. Tubers mixed with condiments are used in arthritis. 141. Habenaria plantaginea Lindl. The Platago-Like Tuber Tablet made form tuber paste 55 Habenaria (E) cure chest pain and stomachache. 142. Habenaria repens Water Spider Orchid Tuber It is used as aphrodisiac 72 Nutt. (E) 143. Habenaria roxburghii Roxburgh’s Tuber Decoction from tuber applied 55 Nicolson Habenaria (E) externally to cure snake bite. 144. Herminium lanceum Jalya (H) Stem, leaves Cures diabetes, fever, 74,75 (Thunb.ex Sw.) bleeding and urinal disorders. 145. Herminium monorchis (Linn.) The musk orchid Roots Tonic 30 R .Br. 146. Hetaeria oblique The Deviating Whole plant The plant is used by Malaya 76 Blume Hetaeria (E) for poulticing sores. 147. Liparis nervosa - Tubers Cure malignant ulcers and (Thunb) Lindl. stomachache 148. Listera ovate Common Twayblade Tubers Used to treat Stomach diseases 77 (L.) R. Br. (E) and bowel irritation. 149. Lusia tenuifolia - Rhizome The rhizomes and leaves are 78 Blume used as an emollient.. 150. Lusia trichorhiza - Root Paste of dried plant cure 17 (Hook.) Blume jaundice, reduce muscular pains and even antidiarrhoea (for cattle). 151. Luisia zeylanica - Stem Stem is used in burns and to 59 Lindl. treat fractures. 152. Malaxis acuminta Rishbhak (H) Rhizome, Used as tonic to cure 2,72 D.Don pseudobulb tuberculosis, fever and enhance sperm production. It is important ingredient of Chayvanprash of ‘Asthavarga’

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) group of drug. 153. Malaxis cylindrostachya Adder Mouth Orchid Pseudobulb Pseudobulb preparation is 2 (Lindl.) Kuntze (E) considered as tonic 154. Maxillaria densa The Crowded Whole plant Painkiller 79 Lindl. Maxillaria (E) 155. Malaxis mucifera Jeevak (H), Adder Pseudobulb It is important ingredient of 2 (Lindl.) Kuntze Mouth Orchid (E) ‘Asthavarga’, used as tonic, rejuvenating drug and cure fever, phthisis. 156. Malaxis rheedii The Resupinate Tuber Paste of tuber is useful in 55 Sw. Malaxis (E) case of insect bite and rheumatism. 157. Malaxis wallichii The Gradually Pseudobulb It is said to cure tuberculosis 4 (Lindl.) Deb Tapering Malaxis (E) and enhance sperm formation. 158. Microstylis mucifera - Root Roots of plant promote -do- (Linn.) Ridl. sperm formation. 159. Neottianthe calcicola - Rhizome Tonic 30 (W.W. Sm.) Soo. 160. Nervilia aragoana Aragoa-Like Nervilia Leaf, tuber Leaves and tuber paste is 55 Guad. (H), Sthalapadma (H) used as ointment and medicine after childbirth 161. Nervillia plicata Pleated Leaf Nervillia Tuber Tuber paste is used as an -do- (Andr.) Schltr. (E) external application for insect bites. 162. Nidema boothii Booth’s Nidema (E) Whole plant Relaxant agent 63 (Lindl.) Schltr. 163. Oberonia anceps - Leaf The malayas use the leaves 76 Lindl. for poulticing. 164. Oberonia caulescens - Tubers Useful in lever disorders. 30 Lindl. 165. Oberonia pachyrachis Thick Spine Oberonia Leaf Antibacterial 37 Rchb.f.ex Hook.f. (E) 166. Oberonia wightiana - Leaf Leaf is crushed and taken as 55 Lindl. medicine to suppress tumour. 167. Orchis latifolia Salep orchid (E) Tuber Tuber powder is added in 21 L. milk or water to treat chest irritation, diarrhoea and

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) chronic dysentery 168. Orchis laxiflora Salab mishri (H) Bulb It is used to cure diarrhea, 6 Lam. bronchitis and convalescence. 169. Otochilus albus - Whole plant Tonic 30 Lindl.

170. Otochilus lancifolius - Pseudobulb Treat dislocated and -do- Griff. fractured bones 171. Otochilus porrectus - Whole plant Treats sinusitis rheumatism -do- Lindl. and even used as tonic 172. Paphiopedilum insigne Slipper orchid or Whole plant Effective against amoeboid 36 (Lindl.) Pfitz. Venus Slipper orchid dysentery. (E) 173. Papilionanthe teres Whole plant Paste is useful to treat 30 (Roxb.) Schltr. dislocated bones 174. Pecteilis susannae Susanna’s Pecteilis Tuber Tubers are used in boils. 2 (L.) Rafin. (E) 175. Peristylus lawii - Tuber Useful in case of insect bites. 55 Wight 176. Phaius tankervilliae Nun’s orchid (E) Pseudobulb, Heals swelling, treats -do- (Alt.) Blume whole plant dysentery and act as pain killer. 177. Pholidota chinensis The Chinese Pseudobulbs Is taken for scrofula, feverish 80 Lindl. Pholidota(E) stomachache, toothache, chronic bronchitis, and duodenal ulcer. 178. Pholidota articulata Harjojan (H), Whole plant Tonic, antibacterial, bone 2 Lindl. The Articulated fracture, skin diseases. Pholidota (E) 179. Pholidota imbricata Rattlesnake Orchid Whole plant The plant is used as tonic, 35 (Roxb.) Lindl. (E) cure abdominal pain, rheumatism and even heals bone fractures. 180. Pholidota pallida The Pale Pholidota Pseudobulb Extracted juice from 81 Lindl. (E) pseudobulb is applied on cut as haemostate. 181. chlorantha The Greater Rhizome Plant is used to make 46

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) (Custer) Rchb. Butterfly-orchid (E) ointment and applied to ulcers. 182. Platanthera sikkimensis - Bulbs, Relieve rheumatic and -do- (Hook. f.) Kraenzlin. Pseudobulb abdominial pain 183. humilis Ground Growing Pseudobulb Heals wound and used as -do- (Sm.) D. Don Pleione tonic 184. Pleione maculate The Spotted Pleione Rhizome Used in stomach and liver 35 (Lindl.) Lindl. (E) disorder 185. Pleione praecox Pseudobulb Used in healing of wound 30 (Sm.) D. Don and used as tonic 186. Polystachya concreta Pale Flowered Tuber Decoction of tuber with honey 17 (Jacq.) Garay & Sweet. Polystachya (E) is useful for treatment of arthritis. 187. Rhynchostylis retusa Banda, Rasna (H), Leaf, whole Antibacterial and cure 2 (L.) Blume Foxtail Orchid (E) plant rheumatic disease. 188. Saccolabium papillosum - Root It has cooling effect and 21 Lindl. specific for rheumatism. 189. Satyrium nepalense Salam misiri, Tuber, root The dried tubers are used in 2 D.Don. Banalu (H) tonic and also in malaria and dysentery 190. livida The Bluish Whole plant Analgesic agent and anti- 79 Schltr. Scaphyglottis (E) inflammatory. 191. Seidenfia rheedii (sw.) Szlach. The Resupinate Root Roots decoction with honey 17 Malaxis (E) cure cholera 192. Smitinandia micrantha - Whole plant Tonic and antibacterial 30 (Lindl.) Holttum 193. Spiranthes australis Pink Spiral Orchid Whole plant Treatment of bacterial and 82 (R.Brown) Lindl. (E) inflammatory diseases, cancer, blood and chest disorder. 194. Spiranthes mauritianum - Whole plant Used for snake bites and 83 Scop. scorpion stings. 195. Spiranthes sinensis Austral Ladies Roots Aphrodisiac, treatment of 84,85 (Pers.) Tresses (E) hemoptysis, epistaxis, headache, chronic dysentery and meningitis. 196. Taprobanea spathulata The Spathulate Vanda Flower, Tuber’s decoction cure 19 (L.) Sperg. (E) whole plant asthma and mania.

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S. No. Botanical Name Common Name Part(s) Used Ethnomedical Uses Reference(s) (E:English, H:Hindi) 197. Thunia alba Whole plant Useful in treating dislocated 30 (Lindl.) Rchb. F. bones. 198. Tropidia curculigoides Lindl. The Curculigo-Like Root Cure diarrhoea and malaria. 86 Tropida (E) 199. Vanda coerulea Autumn lady’s tresses Flower Effective against glaucoma 87 Griff.ex Lindl. orchid, blue vanda (E) and blindness. 200. Vanda cristata The Comb Vanda(E) Leaf Antibacterial and used as 88 Wall. Ex Lindl. expectorant 201. Vanda parviflora The Small Flowered Leaf , root Antiviral, anticancerous and 89 Lindl. Vanda(E) treats nervous disorder, rheumatism etc. 202. Vanda roxburghii Vandae (E) Leaf, roots Brings down fever, treatment 90 R.Br. of otitis, dyspepsia and rheumatism. 203. Vanda spathulata The Spathulate Vanda Flower Flower’s Powder are used in 91 (L.) Spreng. (E) the treatment of asthma and mania 204. Vanda tessellata Vanda (E) Roots, leaves Aphrodisiac, analgesic, 17, 72, 86, 92 (Roxb.) Hook. ex G. Don. and flowers nervine tonic and used in sexually transmitted diseases, fever, rheumatism . 205. Vanda testacea The Brick-Red Vanda Root Cure earache, asthma and 17 (Lindl.) Rchb.f. (E) bone fracture of cattle 206. Vaniila griffithii Griffith’s Vanilla (E) Leaf, stem It provides strength to root of 76 Rchb.f. hair 207. Vanilla planifolia Flat-leaved Vanilla, Fruit Aphrodisiac and main source 35 (Jacks. ex Andrews) Tahitian Vanilla West of vanilla Indian Vanilla (E) 208. Vanilla walkeriae - Stem It is used to cure fever in 93 Wight cattle and nutritive supplement 209. Zeuxine strateumatica Lawn orchid, Tuber Source of tonic and salep. 21 (L.) Schltr. Soldier’s Orchid (E)

The important medicinal orchids are Habenaria intermedia (Riddhi), Platenthera edgeworthii (Vridhi), Malaxis acuminata (Rishbhak), Malaxis muscifera (Jeevak), Dendrobium macraei (Jivanti) and Satyrium nepalense (Salam misiri) 2. The first four of the above-listed species are the components of ‘Asthavarga’ (group of eight medicinal plants) that is a vital part of highly popular polyherbal

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Singh Deepak Kumar, IJSRR 2018, 7(3), 1990-2036 formulation “Chyvanprasha”, widely used as a tonic, rejuvenator, anabolic, immunomodulator and memory enhancer 2,6,50,72. However, the most important medicinal orchid is Dactylorhiza hatagirea (Salam Panja). In addition to the ‘Asthavarga’ orchids, a number of orchids are well known for their significant medicinal properties along with their ornamental merit. Antibacterial activities of Aerides multiflorum and Coelogyne nitida have been reported against Salmonella auereus and Klebsiella pneumonia 4,22, whereas Dendrobium farmeria inhibits the growth of Escherichia coli, Salmonella typhi and Klebsiella pneumonae 4. Recently, methanolic extract of Satyrium nepalense has been shown to possess excellent antibacterial activities against gram (+)ive and gram (-)ive bacteria, namely Staphylococcus mutans, Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiellla pneumonia, which are pathogenic for human beings 94. Distinctive blood clotting attribute of decoction obtained from crushed leaves of Cymbidium giganteum was reported by 55. 17demonstrated that 6-8 drops of decoction of roots of Habenaria commelinifolia administered orally in empty stomach for 10 days cures urinary troubles and spermatorrhoea. Habenaria edgeworthii have shown excellent rejuvenating and disease preventing properties 2. Dried tubers of this species are used to cure skin and cardiovascular diseases. Dendrobium has been used as a source of tonic, antipyretic, astringent, aphrodisiac and anti-inflammatory compounds56, 96. Antioxidant activity and production of phenolic compounds in Habenaria edgeworthii, an important ‘Ashtavarga’ plant, has been confirmed by 97. Singh and Tiwari, (2007) demonstrated therapeutic potential of different Eulophia species for various ailments like fertility, aphrodisiac, anti-rheumatic, antifutigue, skin protective, wound healing and antitumour activity98. The chloroform extract of Cymbidium aloifolium showed significant antibacterial activity against ten potential clinically pathogenic bacteria, viz. Klebsella oxytoca Proteus vulgaris, Pseudomonas aerosinosa, Pseudomonas mirabilis, rah anginosus Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus mitis and Xanthomonas sps. 99. Chinsamy et al., (2014)confirmed anti-inflammatory, antioxidant and anti-cholinesterase activity of seven South African medicinal orchids, viz. Ansellia africana, Bulbophyllum scaberulum Cyrtorchis arcuata, Eulophia hereroensis, Eulophia petersii, Polystachya pubescens and Tridactyle tridentata100. Recent investigation of Bhattacharya et al., (2015) on chemical profiling of in vitro raised plants of Dendrobium thyrsiflorum revealed the presence of different secondary metabolites and significantly higher antioxidant activity of in vitro raised shoots than those from the mother plants101. The therapeutic properties of orchids are due to the presence of secondary metabolites, such as, flavonoids, alkaloids, glycosides and other phytochemicals 2,102,103. Bisbenzylerianin, an active principle isolated from Dendrobium chrysotoxum is an antioxidant 104, whereas ‘habenariol’ an active principle isolated from Habenaria edgeworthii acts as a repellent against herbivores 105. The tuberous

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Singh Deepak Kumar, IJSRR 2018, 7(3), 1990-2036 roots of Eulophia species are rich in bioactive substances, Eullophiol, Ephemeranphol, Fimbriol, Lusianthridin, Nudol, β-Sitosterol and β-Sitosterolglucoside 98. The methanolic extract of Cymbidium aloifolium has been reported to contain alkaloids anthraquinones, flavonoids, simple sugars, tannins, terpenoids, etc. 99 NEED OF TISSUE CULTURE Since long tissue culture techniques have been used for propagation of rare, endangered and threatened orchids 106. A single capsule contains millions of microscopic seeds 11-14. Only about 1% of these are able germinate. If all these had the ability to develop onto plants, entire world would have been full of orchids 107. Rate of germination in orchids is relatively low due to failure of endosperm development and lack of fungal infection. Besides, a skinny cuticle around a small embryo is not adequate to protect it against desiccation 108. In vitro methodologies circumvent these difficulties using which almost all seeds can be made to germinate on simple defined media, containing sugar, under controlled temperature and humidity conditions. Sometimes mutualistic association proves fatal for orchids due to the formation of net like structure around embryo and secretion of harmful substances by fungi and algae 109. To bypass this symbiotic relationship and severe consequences of injurious products, tissue culture is the only method, which provides all the required factors necessary for seed germination and seedling growth 109. Multiplication by mean of vegetative propagation is extremely slow and time consuming to generate large quantity of orchids replica 110. Its slow growing properties hardly fulfill the need of people, market and various pharmaceutical companies 13. Long maturation process even reduces its market value 11. In vitro methodology can reduce the length of time needed for germination and large scale multiplication 110. Consequently, tissue culture technique has wide range of application in micropropagation of orchids and the only approach to save these critical sources of medicine.

METHODS OF ORCHID MICROPROPAGATION Several media have been tested for asymbiotic in vitro germination of orchid seeds. The most commonly used media are MS 111, Mitra’s 112 and Knudson 113. Other media like p723 (PhytoTechnology Orchid seed sowing Medium),114 VW (Vacin and Went Modified Orchid Medium)114, MM (Malmgren Modified Terrestrial Orchid Medium,114 BM-1(Terrestrial Orchid Medium),114 HP (Hyponex peptone medium)115 and NDM (New Dogashima Medium)116 have also been used for in vitro culture of orchids. However, detailed in vitro studies are available only for few genera viz., Acampe, Bletia, Cleisostoma, Cymbidum, Cypripedium, Dactylorhiza, Dendrobium, Epipactis, Eria, Geodorum, Goodyera, Grammaophyllum, Habenaria, Laelia, Malaxis, Oncidium, Paphipedilum, Phalaenopsis, Rhynchostylis, Vanda and Vanilla. These are highlighted in Table 2.

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Seed germination Orchid seeds do not possess enzymes to metabolize polysaccharides and lipids11. Besides, being non-endospermic they lack sufficient reserve food material to support growth of embryo. Therefore, to fulfill this requirement, seeds enter into symbiotic relationships with various mycorrhizal basidiomyceteous fungi to provide required nutrients for their germination14. Fungal mycelia enter the seed, penetrate the germinating embryo and relationship between fungus and seed is established. Endomycorrizal fungi break down starch to release sugar for utilization by the developing embryo. Symbiotic fungus provides the embryo organic material, water and mineral nutrients117. In nature, high proportions of seeds fail to survive and germinate as the mycorrhizal association is not common. To overcome this problem Knudson, (1922) established methodology for asymbiotic seed germination on suitable artificial medium under controlled conditions109. He demonstrated that orchid seeds could germinate on simple nutrient medium containing sugars without mutualistic relationship109. Knudson, (1930) also highlighted that obligate symbiosis was not necessary either for seed germination or for flowering118. Germination potential of embryos varies depends on their developmental stage119. The immature seeds germinate readily and much better than the mature ones. This conclusion is based on studies on in vitro germination of seeds of Cypripedium calceolus, Dactylorhiza maculata, Epipactis helleborine, Goodyera repens, Gymnadenia conopsea120 Dendrobium florum, Cymbidium elagans110, Satyrium nepalense121, Habenaria edgeworthii122, Acampe papillosa123, Dendrobium thyrsiflorum101. On the other hand, some investigators have reported in vitro germination of mature seeds of Phalaenopsis gigantean116, Cymbidium sp.125 was better than immature seeds. During asymbiotic germination, embryo swells to from a spherule which develops absorptive epidermal hairs known as rhizoids126. The embryo is oval shaped with larger cells at basal region and smaller meristematic cell at upper region, whereas in the later stages of development there is formation of achloropyllous and chlorophyllus protocorm like bodies109. The term protocorm was first used by Treub, (1884) during analysis of sporophyte development in lycopodiaceae127. Morel (1960) introduced the term protocorm like bodies (PLB) for protocorms developing in orchid tissue cultures128. Rasmussen, (2002) Considered protocorm equivalent to radical and hypocotyls of seedlings of other plants15. Shape of the protocorm is not constant and these are spherical, oval, round, oboviform, elongated branched, disk, spindle or thorn shaped129-130. Protocorm of Calypso bulbosa are round131, whereas those of Goodyera repen are elongated132. In vitro germination of seeds has been successful in Geodorum densiflorum133, Cypripedium calceolus, Dactylorhiza maculata, Epipactis helleborine, Goodyera repens, and Gymnadenia

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Singh Deepak Kumar, IJSRR 2018, 7(3), 1990-2036 conopsea120, Malaxis khasiana12, Oncidium sp.16, Rhynchostylis retusa134, Dendrobium candidum135, Bletia purpurea114, Satyrium nepalensis121, Laelia speciosa136, Cymbidium elagans, Dendrobium densifolium110, Eria bambusifolia 13, Paphiopedilum sp.137, Cymbidium giganteum42, Habenaria edgeworthii122, Cymbidium aloifolium138, Dendrobium aphyllum14, Phalaenopsis gigantean116, Acampe papillosa123 and Dendrobium thyrsiflorum101. Factors affecting seed germination Non symbiotic seed germination of orchids is greatly influenced by several factors like seed age, different media, PGR, sugar, carbohydrates, vitamins, temperature, light, pH, atmospheric conditions, moisture and orientation of the explants on the medium109,139. In vitro seed germination of mature seeds is generally a difficult task108. Thus, only 13 and 31% of 200 day old seeds of Paphiopedilum villosum var. densissimum germinated on KC medium after 40 and 80 days of culture, respectively137. However, about 70% of mature seeds of Acampe papillosa germinated and differentiated protocorms on being cultured on Mitra’s medium supplemented with coconut water (15%) 123. Choice of sugar used in the culture medium too influences germination of seeds and further growth of seedlings139. Moreover, requirement of sugar varies with different developmental stages of seed germination140. L-glucose and L-mannose failed to support germination of seeds of Cymbidium elagans and Coelogyne puntulate, while other sugars, such as, sucrose, D-glucose, maltose, trehalose and raffinose significantly enhanced germination frequency of these plants of 137,141, which was in accordance with analysis of 142. According to Harvais, (1982) cytokinins are the most important growth regulators for germination of ground orchids143. Arditti and Ernst, (1984) Opined that orchid seeds are more sensitive to higher cytokinin levels than the protocorm144. Deleterious effect of 2,4-D on seed germination and differentiation of protocorm is reported by many authors141,145,146. Pradhan and Pant, (2009) Observed better seed germination in Cymbidium elagans when BAP (1mg/l) along with NAA (0.5mg/l) was added to the medium110. Swar and Pant, (2004)also reported similar results for seed germination of Cymbidium irridioides147. The frequency of germination of seeds of C. irridiodes was the maximum when MS basal medium was used for in vitro seed germination of C. irridoides148. Similar results were obtained by Pant and Gurung, (2005) for Aerides odorata149. Peptone is a water soluble protein with high contents of amino acids, amides and rich in vitamins which stimulate seed germination 150. According to Hossain et al., (2010), seed germination and health of protocorm depends on peptone supplemented to the culture media used for germination of seeds of Cymbidiuim gigantem42. Study made by Curtis, (1947) indicated that very little concentration of peptones (0.05%) proved synergistic to seed germination in Paphiopedilum and Vanda sp151. Recent reports even

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Singh Deepak Kumar, IJSRR 2018, 7(3), 1990-2036 observed that peptone supplemented Mitra’s medium enhanced seed germination in Herminium lanceum and Satyrium nepalense152,153. In contrast, peptone reduced seed germination of Habenaria clalvellata 11. Biotin and nicotinic acid along with pyridoxine promoted seed germination of Orchis laxisflora 154. Sharma et al., (1991) reported that presence of many vitamins in the media favored germination and growth of seedlings of Cymbidium elagans and Coelogyne punctulata155. Depauw et al., (1995) observed synergistic role of BAP with modified barley medium in enhancement of seed germination of Cypripedium spp156. PROLIFERATION AND DEVELOPMENT OF MULTIPLE SHOOTS Role of cytokinins has been variously addressed in in vitro studies on orchids. TDZ is one of the most commonly used growth regulators in orchid tissue cultures. Mahendran and Bai, (2009) reported maximum frequency of multiple shoots (14.62 shoots/explant) formation in Satyrium nepalense on MS medium supplemented with 13.76µM TDZ and it reduced significantly on the same medium containing lower concentration of TDZ (4.52µM) 121. The effectiveness of TDZ in shoot proliferation has also been reported for Anoectochilus formosanus 157, Dendrobium hybrids 158, Dendrobium candidum 159 and Phalaenopsis gigantean 116, Herminium lanceum 153.TDZ has been reported to adversely influence elongation and rooting of regenerated shoots of Pinus strobus(cited in 160. This might be due to its greater persistence power to stay inside tissues in contrast to other adenine type cytokinins, BAP or KN 160 . Huang et al., (2001) found deleterious effects of TDZ on proliferation of shoots and rooting of Paphiopedilum hybrid124. Nayak et al., (1997 a, b) developed an effective protocol for the elongation shoots of Acampe praemorsa, Cymbidium aloifolium, Dendrobium moschatum to circumvent the problem earlier encountered by Huetteman and Preece, (1993) by transferring the shoot clumps regenerated on MS+TDZ to MS +0.5mg/l BAP+2mg/l NAA160-162. TDZ was more successful than rest of the cytokinins in inducing multiple shoots from different explants of Acampe praemorsa 161,162. Rao et al., (1993) observed that number of shoots and leaves of Vanilla planifolia significantly reduced with increasing concentration of BAP163. This is in tune with the findings of Bhatt, (1994), who reported that increasing the concentration of cytokinin proved deleterious for shoot growth of Vanilla planifolia164. Similar results were obtained by Neelannavar et al., (2011), who observed that lower concentration of BAP (1.5 mg/l) than the higher levels in MS medium resulted in more shoots (4.70 per explant) of Vanilla planifolia165. High frequency of shoot formation within four weeks of culture of rhizome sections of Geodorum densiflorum was observed on MS medium fortified with 5µM BAP 166. Apart from cytokinins, Tan et al., (2013) evaluated the effect of sodium nitroprusside on shoot regeneration and multiplication of Vanilla planifolia, where the number of shoots/explant showed a significant increase in the presence of SNP and more than 93% of the explants formed shoots167. In Malaxis acuminata, about 98 percent of pseudobulb segments

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Singh Deepak Kumar, IJSRR 2018, 7(3), 1990-2036 responded positively and formed 11 shoot buds/explant within 5-6 week of culture on MS medium enriched with CH (100 mg/ L), NAA and BAP (6 μM each) 168. In Dendrobium thyrsiflorum, the maximum (96%) regeneration frequency along with an average 17.7 shoots/explants with a mean length of 3.5 cm was observed on MS medium containing 2 mg/l TDZ and 0.5 mg/l NAA 101. Among different auxin-cytokinin combinations, BAP (0.2, 5 mg/l) and NAA (0.1, 0.5 mg/l) promoted shoot organogenesis in four species of Paphiopedilum, P.densissimum, P. insigne, P. bellatulum, and P. armeniacum 137. Likewise, regeneration of plantlets and PLB proliferation in Malaxis khasiana was better on MS medium supplemented with NAA (10 µM) and BAP (8µM) than on MS basal medium 12. Similar results were obtained in Grammatophyllum speciasum169, Oncidium sp. 16, Rhynchostylis retusa 134 and Geodrum densiflorum 133. Contrary to above reports, Dutta et al., (2011) reported that auxin-cytokinin interaction was not beneficial for the proliferation of PLBs into multiple shoot production in Dendrobium aphyllum14. It was possible to induce multiple shoot formation if IAA or KN were used individually. In Malaxis acuminata, all responding explants produced single adventitious shoot irrespective of the type and concentration of the cytokinin, but addition of 0.5mg/l NAA to the medium enhanced adventitious shoot formation 170. PLANT REGENERATION IN CALLUS CULTURES Callus cultures of orchids have shown limited success because of difficulty in callus induction, their limited growth often accompanied with severe necrosis 55,106,171,172. A number of authors feel that initiation and subculture of callus in orchids is challeng 171,172,173,174,175. Nevertheless, several investigators have reported beneficiary role of exogenous auxins in callus production, maintenance and development in a number of orchids,e.g. Cymbidium ensifolium 176, Paphiopedilum hybrid 104, Dendrobium fimbriatum 177, Cymbidium sp. 178,179 Pahiopedilum sp. 180. 181 made an effort for long term callus cultures of Paphiopedilum. Induction of totipotent calli from seed derived protocorms of Cypripedium formosanum (slipper orchid) on 1/2 MS+2,4-D(0-5mg/l)+TDZ(1mg/l) was reported by Lee and Lee, (2003) and Lu, (2004) 182-183. Friable and light yellow callus was induced within eight weeks, when seeds of Habenaria edgeworthii were cultured on MS medium or same fortified with 1 µM NAA. This was sub-cultured repeatedly after four-week intervals to increase its biomass 97. Somatic embryogenesis is not well documented for orchids 175,184. However, Chen et al., (1999) observed the development of somatic embryos on leaf tip explants of Oncidium61. Roy and Banerjee, (2003) and Roy et al., (2007) observed the formation of embryogenic callus from shoot tip explants of Dendrobium fimbriatum and Denrobium chrysotoxum55-177.

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Singh Deepak Kumar, IJSRR 2018, 7(3), 1990-2036 ROOTING, HARDENING AND ACCLIMATIZATION OF PLANTLETS Rooting of in vitro regenerated shoots is a critical step in any micropropagation protocol. The roots developed should be hardy enough to support the plantlets on being transferred from in vitro conditions to ambient ones. Generally, an auxin or rarely a combination of auxins is used for the rooting of shoots of orchids. Hossain et al., (2010) reported development of solid root system from PLBs and shoot buds of Cymbidium giganteum inoculated on half strength of Phytamax or Mitra’s medium supplemented with 0.5mg/l IAA. In Vanilla planifolia, IBA alone at 0.5mg/l proved to be the best in inducing the highest number of roots along with good length in small time 42-165. The efficiency of IBA in root induction has also been observed in Cymbidium pendulum 185. These results are also consistent with the findings of Mohanty et al., (2012), who successfully rooted regenerated shoots of Dendrobium nobile by transferring them to MS medium containing 1.5 mg/l IBA186. Likewise, in vitro shoots of Dendrobium thyrsiflorum rooted best on 1/2 MS medium supplemented with 1 mg/l IBA and 0.5 mg/l phloroglucinol (Bhattacharya et al., 2015) Even the in vitro raised shoots of Satyrium nepalense were rooted on MS medium fortified with 9.84µM IBA 101-121 .Similarly, in Hermnium lanceum best rhizogenic response was observed in 0.1 µM IBA supplemented Mitra’s medium 153. However, IAA and IBA were not always effective in inducing roots in many species of orchids. Sheelavanthmath et al., (2000) reported ineffectiveness of IAA and IBA in induction of roots from shoots of Geodorum densiflorum , 100% of which developed shoots on medium containing NAA (1µM) 166. The combination of NAA with BAP proved to be differentiation of shoots and their rooting in Grammatophyllum speciasum, Oncidium sp., Thynchostylis retusa and Geodrum densiflorum 16,133,134,169. Werckmeister, (1971) first used charcoal to darken the medium for culture of shoot tip derived Cymbidium plantlets187. This was followed by Ernst, (1974 (a,b) 1975) who used it for seed germination of Paphiopedilum and Phalaenopsis188,189,190. Cheruvathur et al., (2010) observed that the presence of activated charcoal was compulsory for root induction in Malaxis acuminata, irrespective of the auxin used170. This could have been due to reduction of light at the base of plants because of the inclusion of charcoal in the medium, thus resulting in reduction of inactivation of photosensitive auxin (IAA) absorption of inhibitory substance, such as, polyphenols (Pan and Staden, 1998), adsorption of high concentration of growth regulator like IAA, NAA, IBA, BA, KN (Weatherhead et al., 1979) and ethylene 191-193. Eymar et al., (2000) observed that AC maintains pH, increases nitrogen uptake, improves growth and reduces inhibitory effect of exogenous cytokinin on rooting194. Piri et al., (2013) too reported formation of root primodia in Acampe papillosa when Mitra’s medium was fortified with AC (2g/l), CW (15%) and YE (2g/l) 123 .The incorporation of lower concentration of activated charcoal (< 0.3%) in the MS medium promoted healthy root formation and pigmentation of the plantlets in

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Malaxis acuminate 168. Critical role of AC in root induction has also been reported in Rananthera imschootiana 195; Anoectochilus formosanus 157 Cymbidium faberi 196 and Dendrobium hybrid 158. Gruenschneder, (1973) reported that AC reduced browning and stimulated root development in Dactylorhiza maculate197. In Cymbidium, it assisted in establishing polarity so that roots become positively geotropic 187. Effective and successful tissue culture can only be realized when plantlets are transferred from in vitro to ambient conditions 198. A wide variety of potting mixes (substrata) have been used for the transfer and acclimatization of in vitro raised orchid plantlets. Giri et al., (2011) reported maximum (87.5%) rooting when elongated shoots were transferred to half strength MS basal medium, where shoots developed tuberous roots after two months of culture122. Nearly 68% survival rate was recorded when shoots of Habenaria edgeworthii with elongated roots were transferred to a mixture of soil:sand:perlite (1:1:1) ratio. Similar results were also obtained for Habenaria bractescens (Medina et al., 2009) and Habenaria macroceratitis 199 -200 . Franco et al., (2007) appraised the effect of ten substrates (pine bark, coco fibre, wood shaving, polystyrene foam etc.) on establishment of in vitro raised plants of Cattleya trianae201. On potting mixes comprising pine:coco:fibre,coal (1:1:1), coco:fibre (1:1) and pine:coco fibre (1:1) 60, 76 and 86%, respectively of the transferred plants survived. The lowest survival (12%) was on pine bark. Rooted shoots of Malaxis acuminata survived well when transferred to a potting mixture of charcoal chips and soil (1:1), covered with polybags and mist irrigated with half strength of MS liquid media 170. The maximum survival (82%) of rooted shoots of Dendrobium nobile was obtained in the compost consisting of charcoal chunks and brick pieces (1:1) with a top layer of moss with 167. Tan et al., (2013) reported 85.0% survival rate after 4 weeks of acclimatization, when in vitro well developed rooted shoots of Vanilla planifolia were transferred to potting mixture having sand and compost (1:2) 186. However, when substratum containing chopped forest litter, coco pits and sand (1:1:1) was used for acclimatization of Malaxis acuminata, 75% survival was observed after 2 month of transfer 168. Recently, in vitro rooted shoot of Satyrium nepalense and Herminium lanceum were successfully hardened in pots having potting mixture of sand and vermiculite(1:1)152-153. A summary of some of the in vitro studies on orchids is provided in Table 2.

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Table 2-Some recent tissue culture studies on orchids. Taxa Type of Medium pH PGR (mg/l) Other Remarks Investigato Cultur Used Adjuste Supplements rs e d Acampe Seed M, - - AC(2g/l) M+CW = maximum 123 papillosa (Lindl.) PDA CW (15%) (70.75%) germination Lindl. YE(2g/l)

Aerides crispum PLB, MS 5.6 BAP (0.1 to Sucrose BAP at 5.0 µM induced 202 Lindl. leaf 1.12), (2%) multiple shoots N (0.1 to 1), Agar (1%) TDZ (0.1 to 1.1), AA (0.08 to 0.8), AA (0.09 to 0.9) Bletia Seed KC, ½ 5.8 - Sucrose Photoperiod stimulated 114 purpurea(Lam.) MS, (2%) seed germination DC. BM-1, MM, VW, P723 Cleisostoma Seed MS,M, 5.6 NAA (0 to Sucrose MS+IAA (1.7 mg/l) + 12 racemiferum(Lin KC 5.6), (3%) BA (1.8 mg/l) = multiple dl.) Garay BA (0 to Agar (0.8%) plantlet 0.2), MS+NAA (1.8 mg/l) + IAA (0 to KN (1.9 mg/l) = well 3.5), differentiated root KN (0 to 3.9), Cymbidium Seed MS,M, 5.6 BA,TDZ,KN Sucrose MS+NAA(0.5 138 aloifolium (L.) KC (0 to 2), (2%) mg/l)+BA(0.1 mg/l) = IAA, NAA Agar (0.8%) 90% seed germination (0 to 1.6) Cymbidium Seed MS 5.8 BAP (0.5 to Sucrose MS+BAP (1 mg/l) = 110 elegans Lindl, 2), (3%) Agar Stimulated seed .Dendrobium NAA (0.5) (0.8%) germination densiflorum Lindl. ex Wall.

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Taxa Type of Medium pH PGR (mg/l) Other Remarks Investigato Cultur Used Adjuste Supplements rs e d Cymbidium, Pseudo MS, ½ - TDZ (0.1 to Sucrose Different concentration of 203 Epidendrum, bulb, MS 1), (4%) NAA and TDZ formed Oncidium, rhizom 2,4-D (1 to embryos and maintained Paphiopedilum es, 10), platelets development and Roots NAA (0.1 to Phalaenopsis. 0.5), BAP (5) Cymbidium Seed MS, 5.8 BAP (0 to Sucrose M/PM+peptone(2g/l)+B 42 giganteum Wall. KC, 2), Agar (0.8%) AP(1mg/l) = 100% seed ex Lindl PM, M IAA, 2,4-D AC (2%) germination (till 2) Peptone M/PM+AC = largest (2g/l) PLB Cypripedium PLB ¼ MS, 5.5 NAA (till Sucrose HP with NAA and 115 macranthos var. HP 0.5), (2%) cytokinin proved best for rebunense BAP( till Agar (0.6%) PLB proliferation 0.22), Zeatin(2.2) Dendrobium Seeds MS 5.8 IAA (0 to IAA(0.5 mg/l) = 14 aphyllum 0.5), - maximum shoot length (Roxb.) KN (0 to 0.5) Dendrobium Seed MS, ½ 5.8 KN (0 to Sucrose MS+BAP(1.98mg/l) = 135 candidumWall. MS 2.9), (2%) highest callus induction ex Lindl. BAP (0 to Agar (0.6%) 5), NAA (0 to 1), 2,4-D (0 to 3) Dendrobium Shoot M 5.8 TRIA Sucrose Effective range of TRIA 204 nobile Lindl. tip (1 to 5 (3%) is 2-7 µg/l mcirogram/l) Agar (0.8%) Dendrobium Seed, MS 5.8 BAP, KN, Agarose MS+TDZ(2mg/l)+NAA( 101 thyrsiflorum nodal TDZ (0.8%) 0.5mg/l) = maximum 17.7 Rchb.f segme (each 0 to shoots proliferated nt 4mg/l)+ NAA(0.5mg/

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Taxa Type of Medium pH PGR (mg/l) Other Remarks Investigato Cultur Used Adjuste Supplements rs e d l) Eria Seed MS, KC 5-5.8 NAA, BA, - MS+IAA(2 mg/l) = 13 bambusifolia KN, GA3 enhanced shoot length, Lindl. (0.5,1,2) MS+NAA(2 mg/l) = Best rooting Geodorum Seed MS, ½ 5.4-5.8 NAA (till 2), Sucrose NAA(2 mg/l)+BAP(2 133 densiflorum MS BAP (1 to (3%) mg/l) = enhance (Lam.) Schltr. 2.5), Agar (0.8%) elongation IAA (1), IAA(1 mg/l) = root Zeatin(1), system developed Grammatophyllu PLB MS, ½ 5.7 NAA, BAP Sucrose MS+NAA(2mg/l)+BAP 169 m MS (0 to 2) (2%) (1mg/l) = Optimum shoot speciosum formation Blume Habenaria Multi MS 5.5 BAP (1 to Sucrose 87.6 BAP at 10mg/l 199 bractescens modal 10) milli molar stimulated root tuber Lindl. stem Agar formation (0.65%) Habenaria MS, ½ 5.6 NAA (0 Agar (0.8%) NAA (1µM) = max seed 122 edgeworthii MS to.09) germination’ Hook.f. ex BAP, IBA (0 BA+NAA = maximum Collett to 0.1) shoot Habenaria Shoot ½ MS 5.6 BAP, NAA Sucrose 1/2MS+BAP(0.54 205 radiata(Thunb.) apex (3%) µM+NAA(4.44 µM) = K. Spreng and Agar (0.8%) highest (5.4) adventitious leaf bud/ floret Laeliaspeciosa( Seed KC, MS - BAP (0 to Sucrose MS+NAA(0.5 136 HBK) Schltr. ,1/2 MS 0.5), (3%) mg/l)+GA3(0.1 mg/l) = GA3 (till effective for germination 10), NAA(0 to 1) Malaxis Pseud M 5.7 BAP(1), Sucrose BAP+NAA(1mg/l each) 206 acuminata D. obulb NAA(1) (2%) promoted PLB Don Agar (0.9%) proliferation and plantlet development Malaxis Intern MS 5.8 BAP, KN, Sucrose NAA(0.5mg/l) = 170 acuminata ode TDZ (each 1 (3%) Enhanced adventitious

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Taxa Type of Medium pH PGR (mg/l) Other Remarks Investigato Cultur Used Adjuste Supplements rs e d D.Don to 4) , NAA Agar (8%) shoot, TDZ(3mgl/l) = (0.5) 96% organogenesis Malaxis Pseud MS 5.6 NAA, BAP Sucrose CH+NAA+BAP (6 168 acuminata D. obulb (0 to 9 µM) (3%) µMeach ) = induced Don CH 11shoot bud/explant (100mg/l) after 6week Citric acid (100mg/l) AC(0-0.4%) Malaxis Seed MS, M 5.6 IAA (0 to 1), Sucrose MS+IAA(1 mg/l)+BA(4 12 khasianaSoland BAP (0 to (2%) mg/l)+KN = induced ex. Swartz .07), Agar (0.7%) multiple shoot KN (0 to AC (0.1%) 5.8), NAA (till 0.5) Oncidium sp. Seed MS 5.6 BAP, NAA Agar (0.8%) BAP(2)+NAA(1.5 mg/l) 16 (0 to 4) = 100% shoot and root forming capacity Paphiopedilum Seed MS 5.8 BAP(1 to 8), Sucrose NAA (0.5,0.1), BAP (4, 137 species KN (0.1), (2%) 5.5 mg/l) = maximum NAA ( 0 to Agar (0.6%) shoot were observed 1), TDZ (till 0.5) 2,4-D (1 to 8), IAA, IBA Phalaenopsis PLB, MS, - BAP, KN, Sucrose TDZ with NAA was 116 gigantea leaf, NDM NAA, TDZ (2%) found to be best for PLB ripe (0 to 1) and callus induction capsul e seed Rhynchostylis Seeds MS 5.8 BAP (0 to Sucrose BA(1.3 mg/l)+NAA(0.03 134 retusa Blume 1.8), (3%) mg/l) = Seedling growth NAA (0 to Agar (0.8%) maximum 0.8), AC (0.1%) TDZ(0.44 mg/l) =

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Singh Deepak Kumar, IJSRR 2018, 7(3), 1990-2036

Taxa Type of Medium pH PGR (mg/l) Other Remarks Investigato Cultur Used Adjuste Supplements rs e d IBA (0 to multiple shoot 1.2), TDZ (0 to 1.3), KN (0 to 1.3) Satyrium Seeds MS,KC, 5.6-5.8 IBA, BAP, Sucrose TDZ(13.76 µM) = 121 nepalense D. KCM KN (3%) Multiple shoot, Don (each 1 to 4) Agar (0.8 %) IBA(9.84 µM)= best TDZ (0.5) rooting Vanilla Node MS - BAP (0.1 to Sucrose BAP(1 mg/l)+KN(1 207 planifolia 3), (3%) mg/l) = maximum shoots Andrews NAA (0 to Agar (0.8%) were obtained after 45 1), days KN (0 to 1.5) Vanilla Shoot MS - BAP (0.5 to - BAP(1 mg/l) = enlarged 165 planifolia Andr. tip, 3) shoot intern BAP(0.5 mg/l) = odes, enlarged root leaf segme nt, bud , root Vanilla Node MS 5.8 SNP (0to Agargel 10 µM SNP+ BAP(1 167 planifolia 40µM) (0.55%) mg/l) = highest number Andrews of shoots

Vanda testacea Leave M 5.7 KN, NAA, Sucrose BAP(1 mg/l)+NAA(1 206 (Lindl.) Reichb. s BAP (1) (2%) mg/l) = PLB f. proliferation AC: Activated charcoal, BAP: 6-Benzylaminopurine, CH: Casein hydrosylate, 2, 4-D: 2, 4-Dichlorophenoxyacetic acid,

GA3: Gibberellic acid, IAA: Indole- 3-acetic acid, IBA: Indole-3-butyric acid, KC: Knudson C medium (Knudson C 1946), KN: Kinetin, M: Mitra medium (Mitra et al 1976), MM: Malmgren Modified Terrestrial Orchid Medium, MS: Murashige & Skoog,s medium (Murashige & Skoog 1962), NAA: α-Naphthalene acetic acid, PGRs: Plant growth regulators, PLBs: Protocorm like bodies, SA: Syringic acid, SNP: Sodium nitroprusside, TDZ:-1-Phenyl-3-(1,2,3-thiadiazol-5-yl)-urea , VW: Vacin & Went Modified Orchid Medium

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Singh Deepak Kumar, IJSRR 2018, 7(3), 1990-2036

SYMBIOTIC SEED GERMINATION The presence of fungi in orchid roots under natural conditions was first observed by Reissek, in 1847) 208. Later, in 1866, Wahrlich identified and described various changes taking place in orchids roots due to fungal infection209. Magnus, (1900) was the first to observe peloton inside the cells and even described various stages in fungal colonization210. Recent report by Singh et al., (2017) even confirmed the presence of pelotons in cortical cells of root section of Herminium lanceum and Satyrium nepalense211. Seeds of orchid are shed when embryos are at few celled stage and rest of its development takes place during germination 212. Orchid seeds are extremely small and lack sufficient reserve food material to support the growth of embryo. It depends on mycorrhizal association for the nutrition required by the immature embryo to develop into a protocorm. In most of the species, symbiotic association is established by infecting suspensor cell at the base of embryo, whereas in few cases infection occurs through rhizoids 213. The embryo or protocorm attract symbiotic fungi by producing chemotrophic substances 214,215. The fungal associates convert insoluble carbohydrates to simple soluble form and thereby, provide organic carbon to the developing embryos 216,217. The mycorrhizal fungi are also known to supply nitrogenous compounds(Cameron et al., 2006; Burgeff ,1936; Dijk, 1990), soluble phosphate (Smith, 1967; Alexander et al., 1984) and vitamins (Hijner and Arditti, 1973) during seed germination218-223. There are many studies reporting increase in percentage of seed germination and protocorm development if seeds were cultured along with fungal isolates. Clements et al., (1986) reported that fungal isolates Tulasnella sp. and Ceratobasidium stimulated seed germination in Orchis sp. and Dactylorhiza elata, respectively224. Generally, during symbiotic seed germination continuous exposure in dark is required 225. However, Zettler and Mclnnis, (1994) noticed a synergistic effect of 16 hr photoperiod, during the first 7 days of inoculation, in endangered terrestrial orchid Platanthera integrilabia226. Zettler, (1997) applied symbiotic seed germination technique for the conservation of terrestrial orchids, Platanthera spp. (P. cristata, P. integrilabia, P. clavellata), Spiranthes odorated227. In the presence of fungal associates 3 % seeds of these taxa germinated within two weeks. Stewart and Zettler, (2002) observed that the percentage of seed germination in Habenaria quinqueseta, Habenaria macroceratitis and Habenaria repens increased to 18.1, 50.8 and 55.1%, respectively from less than 1% in controls, after incubation with Ceratorhiza isolates228. Athipunyakom et al., (2004) isolated Epulorhiza repens and Rhizoctonia globularis form the roots of Spathoglottis plicata229. In the cultures of the seeds of the same plant inoculated with these fungi, the percentage of seed germination recorded after 127 day of culture were 42.8% (E. repens) and 12.5% (R. globularis), as opposed to the total absence of germination in control. There is an obscure liaison between orchids and endophytic fungi that whether the fungi isolated form adults plant are also crucial

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Singh Deepak Kumar, IJSRR 2018, 7(3), 1990-2036 for their seed germination 225. Chutima et al., (2011) evaluated effect of endophytic fungi on seed germination of Pecteilis susannae. Seed germination enhanced significantly from 62.1% in controls to 79.9% when cultured along with fungal isolates, Epulorhiza sp230. CONCLUSIONS Present review has made an effort to bring together all possible literature of in vitro propagation of orchids via seeds, rhizomes, shoot tips, internodes, pseudobulbs, PLBs, leaves, roots, node as explants (Table 2). Orchids are rich in demand especially in the field of horticulture due to their splendid glamorous long lasting flower. The protocols already developed can possibly be used for large scale mass multiplication along with ex vitro establishment of rare, threatened and endangered orchids to meet the horticultural and floricultural market demand. Cost effective protocol by using minimal media and inexpensive substitutes such as gelling agent, sugar source and concentration, vitamins etc. need to be developed to facilitate commercialization and conservation programs. Taking threat into consideration, orchids have been placed in Appedix II and some have been included in even Appendix I of Convention on International Trade in Endangered Species of Flora and Fauna (CITES). Therefore it is the most imperative responsibility of human being to save these critical sources of medicine for human welfare.

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38. Zhang D, Liu GT, Shi JG and Zhang JJ. Effects of Coeloglossum viride var. bracteatum extract on memory deficits and pathological changes in senescent mice. Basic Clin Pharma Toxicol. 2006; 98: 55-60. 39. Lin CC and Namba T. Historical and herbological studies on the traditional Japanese and Chinese crude drugs. On the “Shan-ci-gu”. Yakushigaku Zasshi. 1985; 20: 88-98. 40. Watanabe K, Tanaka R, Sakurai H, Iguchi K, Yamada Y, Hsu CS, Sakuma C, Kikuchi H, Shibayama H and Kawai T. Structure of cymbidine A, a monomeric peptidoglycan-related compound with hypotensive and diuretic activities, isolated from a higher plant, Cymbidium goeringii (Orchidaceae). Chem Pharm Bull. 2007; 55: 780-783. 41. Duggal SC. Orchids in human affairs (a review). J Pharm Biol. 1971; 11: 1727-1733. 42. Hossain M, Sharma M, Teixeira da Silva JA and Pathak P. Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl. Sci Hortic. 2010; 123: 479-487. 43. Fossen T and Ovstedal DO. Anthocyanins from flowers of orchids chimaera and Dracula cordobae. Photochemistry. 2003; 63: 783-787. 44. Shimura H, Matsuura M, Takada N and Koda Y. An antifungal compound involved in symbiotic germination of Cypripedium macranthos var. rebunense (Orchidaceae). Phytochemistry. 2007; 68: 1442-1447. 45. Grieve M . A Modern Herbal: the Medicinal, Culinary, Cosmetic, and Economic properties, Cultivation, and Folklore of Herbs, Grasses, Fungi, Shrubs, and Trees with All Their Modern Scientific Uses. Tiger Books International: London: 1998. 46. Dash VB . Materia Medica of Tibetan medicine. Sri Satguru Publication: Delhi: India: 1994. 47. Venkateswarlu S, Raju MS and Subbaraju GV. Synthesis and biological activity of Isoamoenylin, a metabolite of Dendrobium amoenum. Biosci Biotechnol Biochem. 2002; 66: 2236-2238. 48. Yang L, Wang Z and Xu L. Simultaneous determination of phenols (Bibenzyl, phenanthrene, and fluorene) in Dendrobium species by high-performance liquid chromatography with diode array detection. J Chromatogrh. 2006; 1104: 230-237. 49. Wu HS, Xu JH, Chen LZ and Sun JJ. Studies on anti-hyperglycemic effect and its mechanism of Dendrobium candidum. Zhongguo Zhong Yao Za Zhi 2004; 29: 160-163. 50. Man LY, Yan WH and Qing LG. Erianin induces apoptosis in human leukemia HL-60 cells. Acta Pharmacol Sin. 2001; 22:1018-1022. 51. Fan C, Wang W, Wang Y, Qin G and Zhao W. Chemical constituents from Dendrobium densiflorum. Photochemistry. 2001; 57: 1255-1258.

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